Igs7^{tm211(tetO-ChrimsonR/tdTomato,CAG-tTA2)Tasic}

The targeting vector contains (from 5' to 3') a Bxb1 attL site, a partial green fluorescent protein sequence (non-functional 500bp fragment to increase the space between the Bxb1 attL site and the insulator sequence to improve stability of the targeting vector), two copies of chicken beta-globin HS4 insulator element (to reduce reporter gene expression in absence of transactivators), a Tet response element/promoter (tetO), a loxP-flanked STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-PGKpA), ChrimsonR-tdTomato (ChrR-tdT) sequence with a Kv2.1 sequence at the 3' end followed by a 6xHis tag, T7 tag and XPress tag sequence, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability), a BGH polyA, two copies of chicken beta-globin HS4 insulator element, a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG; from pCAGGS), a FRT-flanked STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-TKpA), an internal ribosome entry site (IRES) sequence, a synthetic modified tetracycline-regulated transactivator gene (tTA2(S)), a WPRE, a BGH polyA, a PhiC31 attB site, a PGK-5'hygro cassette, an mRNA splice donor, a Bxb1 attR site, SA, 3' hygro-SV40pA, and PhiC31 attP. ChrR-tdT is the result of a ChrimsonR coupled to the fluorescent reporter tdTomato. ChrimsonR (ChrR) is a light-gated cation channel is optimally excited by light of approximately 590nm wavelength (aka, red-shifted). ChrimsonR is an artificial channelrhodopsin derived by introducing the K176R mutation into the alga-derived channelrhodopsin Chrimson. (J:101977)